Human Molecular Genetics, 2000, Vol. 9, No. 13 1967-1976
© 2000 Oxford University Press
Creation of a mouse model for non-neurological (type B) Niemann–Pick disease by stable, low level expression of lysosomal sphingomyelinase in the absence of secretory sphingomyelinase: relationship between brain intra-lysosomal enzyme activity and central nervous system function
Departments of Medicine and Anatomy and Cell Biology, Columbia University, 630 West 168th Street, New York, NY 10032, USA, 1Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA, 2Berlex Biosciences, Richmond, CA 94804, USA and 3Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
Received 31 March 2000; Revised and Accepted 16 June 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Most lysosomal storage diseases result in neurodegeneration, but deficiencies in the same enzymes can also lead to syndromes without neurologic manifestations. The hypothesis that low levels of residual, intra-lysosomal enzymatic activities in the central nervous system (CNS) are protective has been difficult to prove because of inconsistencies in assays of tissue samples. Experimental correction of lysosomal enzyme deficiencies in animal models suggests that low-level enzymatic activity may reduce CNS pathology, but these results are difficult to interpret owing to the partial and transient nature of the improvements, the presence of secretory hydrolases, and other confounding factors. Using a novel transgenic/knockout strategy to manipulate the intracellular targeting of a hydrolase, we created a mouse that stably expresses low levels of lysosomal sphingomyelinase (L-SMase) in the complete absence of secretory sphingomyelinase (S-SMase). The brains of these mice exhibited 11.5–18.2% of wild-type L-SMase activity, but the cerebellar Purkinje cell layer, which is lost by 4 months of age in mice completely lacking L- and S-SMase, was preserved for at least 8 months. The L-SMase activities in other organs were 1–14% of wild-type levels, and by 8 months of age all peripheral organs had accumulated sphingomyelin and demonstrated pathological intracellular inclusions. Most importantly, L-SMase-expressing mice showed no signs of the severe neurologic disease observed in completely deficient mice, and their life span and general health were essentially normal. These findings show that stable, continuous, low-level expression of intra-lysosomal enzyme activity in the brain can preserve CNS function in the absence of secretory enzyme or other confounding factors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lysosomal storage diseases often present as acute, subacute or chronic forms, depending on the degree of central nervous system (CNS) dysfunction (1). For example, Niemann–Pick disease, which arises from a deficiency in acid sphingomyelinase (SMase), occurs in at least two forms. Type A Niemann–Pick disease presents with hepatosplenomegaly and nervous system involvement in early infancy and death usually occurs by 3 years of age as a result of neurodegeneration (2). In contrast, type B Niemann–Pick disease often presents with hepatosplenomegaly in late childhood or adulthood, but with no or only mild signs of CNS dysfunction (2). Intermediate forms of the disease have also been described (2).
A major goal in the understanding of lysosomal disorders has been to elucidate the metabolic and biochemical basis for neurological sparing in some cases but not others (3). Measurements of SMase activity in extracts of cultured fibroblasts from Niemann–Pick patients have shown only a weak correlation with clinical phenotype (<5% of the wild-type SMase activity in type A versus 2–10% in type B) (2). One study looking at sphingomyelin (SM) degradation by living, cultured fibroblasts from type A and B patients has shown a somewhat greater correlation [1–3% hydrolysis in type A (n = 9) versus 10–60% in type B (n = 6)] (4), and similar in situ enzyme assays in fibroblasts from patients with Niemann–Pick disease and other lysosomal storage diseases have provided partial but inconsistent correlates with disease severity (5–7). None of these studies, however, was able to address the most critical issue, namely the relationship between brain intra-lysosomal enzyme activity in vivo and the absence or presence of neurological symptoms.
Animal models of lysosomal storage diseases have been used to address this issue, but uncertainty still remains. In many of these animal models, enzymatic activity is absent from the brain and neurodegeneration develops. Bone marrow transplantation, enzyme replacement or gene therapy procedures improve neurological function in some of these animals, but the improvement is transient and usually partial (8,9). In the case of intravenous administration of enzyme, small improvements in CNS function have been observed when the therapy is initiated in newborn animals, i.e. before complete establishment of the blood–brain barrier (10; S.R.P. Miranda, X. He, C.M. Simonaro, S. Gatt, A. Dagan, R.J. Desnick and E.H. Schuchman, submitted). Bone marrow transplantation and hematopoietic stem cell-mediated gene therapy experiments in several model systems have shown that when small numbers of donor-derived bone marrow cells migrate across the blood–brain barrier and become established in the CNS as microglia, partial improvement in the CNS disease can occur (11,12; S.R.P. Miranda, S. Erlich, V.L. Friedrich Jr, S. Gatt and E.H. Schuchman, submitted). Similarly, partial but often transient improvements in the CNS disease have been observed in mouse models following direct injection of enzyme-producing cells or viral vectors into the CNS (13–15). In each of these cases, the mechanism of neurological correction almost certainly involves endocytosis by neuronal cells of secretory forms of lysosomal enzymes, but other mechanisms, such as cell–cell enzyme transfer and catabolism of extracellular storage material by non-neural cells, have been proposed (8,9). Moreover, in bone marrow transplantation and gene therapy studies, interpretation of the results is difficult because the mice are often subjected to high levels of radiation or viral infection, which may disrupt the blood–brain barrier and/or cause infiltration of immune cells into the CNS (9). Thus, even in these cases, the relationship between brain intra-lysosomal enzyme activity per se and neurological function has not been definitively determined.
In this context, an animal model of a lysosomal storage disease in which varying amounts of the intra-lysosomal enzyme would be permanently restored in the complete absence of the secretory form and other potential confounding factors would be useful in assessing the relationship between intrinsic brain lysosomal enzyme activity and neurological function. Because the lysosomal and secretory forms of acid hydrolases arise from the same protein precursor (16), however, creation of a mouse model lacking the secretory form would require manipulation of the intracellular trafficking of lysosomal enzymes in the whole animal. In the case of acid SMase (ASM), the secretory form (S-SMase) and the lysosomal form (L-SMase) arise from a single gene, called the ASM gene, which gives rise to a single mRNA and a single mannosylated protein precursor (17,18). SMase precursor molecules destined to become L-SMase are acted on by the cis-Golgi enzyme N-acetylgluosaminyl 1-phosphotransferase and N-acetylgluosamine phosphodiesterase, which results in 6-phosphorylation of some of the mannose residues. After transport of the mannose-phosphorylated SMase to the trans-Golgi, vesicles bearing mannose 6-phosphate receptors shuttle the enzyme into the endosomal/lysosomal system, where it functions to hydrolyze lysosomal SM (18). Those precursor molecules destined to become S-SMase somehow escape mannose phosphorylation in the cis-Golgi and thus do not get shuttled to endosomes/lysosomes. Rather, the distal mannose residues are removed, new ‘complex’ sugars are added and the enzyme is secreted (18). Thus, L-SMase and S-SMase differ in both their final destination and their oligosaccharide structure; in addition, the first six amino acid residues of L-SMase are removed by proteolysis when the enzyme enters lysosomes (18). Finally, although both L-SMase and S-SMase are zinc metalloenzymes, L-SMase acquires Zn2+ inside the cell and thus does not require exogenous Zn2+ in enzyme assays, whereas S-SMase is mostly or partially sequestered from cellular Zn2+ and thus requires extracellular Zn2+ for full activity (18). Both L-SMase and S-SMase are deficient in patients with Niemann–Pick disease and completely absent in ASM knockout (ASMKO) mice (17,18).
By genetically manipulating the ASMKO mouse, which is a model of the severe neurological form (type A) of Niemann–Pick disease (19,20), we have created a mouse with complete absence of the secretory form of acid hydrolase and stable, partial restoration of the intra-lysosomal form. This unique mouse model was created by replenishing ASMKO mice with a transgene encoding a chimeric protein that consists of SMase linked to the 33 amino acid lysosomal targeting peptide of lysosome-associated membrane protein-1 (Lamp1). This targeting motif is composed of a single transmembrane domain and a short cytoplasmic tail containing a tyrosine-based G-Y-X-X-I sorting signal that is both necessary and sufficient to mediate direct transport of a protein from the trans-Golgi network to the late endosome/lysosome compartment (21–24). This motif can be used to target heterologous proteins to lysosomes in transfection studies using cultured cells (21,24).
In this report, we show that ASMKO mice expressing the chimeric SMase–Lamp1 protein have L-SMase, but no S-SMase, activity. These animals have 10–20% of wild-type brain L-SMase activity, 1–14% of visceral L-SMase activity and eventual SM accumulation in all tissues examined, yet they have no clinical or pathological signs of the neurological disease that affects ASMKO mice. These findings are the first to show that stable, low level expression of intra-lysosomal enzyme activity in the brain can maintain normal CNS function in the absence of secretory enzyme or other confounding factors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Creation of ASMKO mice expressing an ASM–Lamp1 chimera transgene
To create mice deficient in S-SMase but having L-SMase, ASMKO mice were crossed with transgenic mice expressing a chimeric ASM protein that contained the potent lysosomal targeting motif of the lysosomal membrane protein Lamp1 (21–24). The transgenic mice were created using the transgene shown in Figure 1A (see Materials and Methods). This transgene was tested initially in transfected CHO cells: L-SMase activity in the transgene-transfected cells was 53 ± 2% higher than in mock-transfected cells, but there was no increase in S-SMase activity in the conditioned medium of the transgene-transfected cells (data not shown). Thus, the chimeric transgene successfully increased L-SMase activity in a transfected cell without increasing S-SMase activity. Using this construct to create transgenic mice, we obtained two founders (Tg14 and Tg19); each founder was serially mated with ASMKO mice, resulting in two separate transgenic lines on the ASMKO background (ASMKO/ChimTg-14 and -19). Genotypes of all mice were verified as described in Materials and Methods and Figure 1B.
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ASMKO/ChimTg mice have a complete absence of S-SMase but have functional L-SMase
To determine whether the ASMKO/ChimTg mice still lacked S-SMase despite the presence of the chimeric transgene, we took advantage of the fact that zinc-dependent S-SMase activity can be measured in serum (25); the source of serum S-SMase is probably apical secretion from endothelial cells (26,27). Thus, serum derived from tail blood was assayed from wild-type, ASMKO and ASMKO/ChimTg mice (Fig. 2). Serum from wild-type mice demonstrated substantial SMase activity above the no-serum control and this activity was ~90% dependent on exogenous Zn2+ (25, and data not shown). As expected (17), ASMKO mice demonstrated no serum S-SMase activity above the control value. Remarkably, serum from both lines of ASMKO/ChimTg mice demonstrated no detectable S-SMase activity above the no-serum control.
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To further examine SMase activity in ASMKO/ChimTg mice, peritoneal macrophages from wild-type, ASMKO and ASMKO/ChimTg mice were assayed for SMase activity. Consistent with our previously published data (17), the conditioned medium of wild-type peritoneal macrophages contained substantial SMase activity, whereas medium from ASMKO macrophages had no activity above the no-enzyme control (Fig. 3A). Furthermore, the activity in the wild-type conditioned medium was ~90% dependent on Zn2+ (17), indicating the presence of S-SMase rather than leakage of L-SMase. Consistent with the serum data (above), the conditioned medium from ASMKO/ChimTg-19 had no SMase activity above the ASMKO value or medium unexposed to cells. To assess L-SMase, we examined cell homogenates (Fig. 3B). There was substantial activity in the wild-type macrophage homogenate and this activity was not dependent on Zn2+ (17), indicating L-SMase. Macrophages from ASMKO mice contained no detectable L-SMase activity, as previously reported (17), but macrophages from ASMKO/ChimTg-19 mice had a level of activity that was ~20% of the wild-type level. These data clearly indicate that ASMKO/ChimTg mice have a complete absence of S-SMase but partial repletion of L-SMase.
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To complement these biochemical data, we conducted an immunofluorescence experiment in peritoneal macrophages comparing the intracellular location of wild-type and chimeric SMase versus the lysosomal enzyme cathepsin D (Fig. 4). In wild-type macrophages, SMase (Fig. 4A) and cathepsin D (Fig. 4B) co-localized with a perinuclear distribution, consistent with localization in lysosomes. The same pattern was obtained in macrophages from ASMKO/ChimTg-19 mice (SMase, Fig. 4C; cathepsin, Fig. 4D). These data clearly demonstrate that chimeric SMase is targeted to lysosomes in vivo.
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L-SMase activity and SM mass in various tissues of wild-type, ASMKO and ASMKO/ChimTg mice
To determine the efficacy and range of chimeric transgene expression in ASMKO/ChimTg mice, brain, liver, lung, heart, kidney and spleen were extracted from 4-month-old wild-type, ASMKO and ASMKO/ChimTg mice and tested for both cellular L-SMase activity and SM content. As shown in Table 1, the levels of SMase activity in these tissues ranged between 1.3 and 18.2% of wild-type values. Brain L-SMase activities were 18.2 and 11.5% of wild-type levels in ASMKO/ChimTg-19 and ASMKO/ChimTg-14 mice, respectively.
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The SM mass in the six organs of 4- and 8-month-old wild-type, ASMKO and ASMKO/ChimTg-19 mice are shown in Figure 5. As expected (19,20,28), all tissues of the ASMKO mice had 4- to 6-fold higher levels of SM than those of the wild-type mice. In contrast, the SM content in the brain and heart of 4-month-old ASMKO/ChimTg-19 mice were indistinguishable from wild-type values and the levels of SM in the liver, lung, kidney and spleen were intermediate between those in wild-type and ASMKO mice. Interestingly, the SM mass in kidney from 4-month-old ASMKO/ChimTg-14 mice, which had an SMase activity of 10.6% of wild-type (versus 1.3% for Tg-19), was 89.6 ± 12.7 nmol SM/mg protein (versus 160 nmol SM/mg for Tg-19 kidney). At 8 months of age, all of the six organs had a higher level of SM mass than the wild-type mass. The brain of ASMKO/ChimTg-14 mice, which had 11.5% of wild-type SMase activity versus 18.2% for ASMKO/ChimTg-19 mice (Table 1), had 30% more SM mass compared with Tg-19 brain at 8 months (71.1 ± 4.3 versus 55.2 ± 6.5, P = 0.05). Thus, low levels of SMase expression prevented SM accumulation in brain and heart at 4 months, but by 8 months of age SM accumulation was observed in all tissues, including the brain.
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Histological examination of tissues from wild-type, ASMKO and ASMKO/ChimTg mice
The cerebella of ASMKO mice lack the Purkinje cell layer, which is the pathological correlate of the ataxia that develops in these mice (19,20,28). This point is clearly demonstrated by comparing the ASMKO cerebellar section in Figure 6B with the wild-type section in Figure 6A. Remarkably, the Purkinje cell layers in the cerebella of both 4- and 8-month-old ASMKO/ChimTg-19 mice appeared completely intact (Fig. 6C and D).
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The livers of ASMKO mice show characteristic intracellular inclusions representing the accumulation of lysosomal SM (19,20,28). As shown in Figure 7, these inclusions were quite prominent in the liver section of a 4-month-old ASMKO mouse (compare Fig. 7B with the wild-type liver section in Fig. 7A). Liver from 4-month-old ASMKO/ChimTg-19 liver (Fig. 7C) had many fewer intracellular inclusions than ASMKO liver, but inclusions were quite prominent in 8-month-old ASMKO/ChimTg liver. In spleen, some inclusions were seen in the 4-month-old ASMKO/ChimTg mice (Fig. 7G), but these became more prominent at 8 months (Fig. 7H). Finally, the lungs of ASMKO mice show partial alveolar obstruction (Fig. 7J), which was also observed, though to a lesser degree, in the lungs of 4- and 8-month-old ASMKO/ChimTg mice. In summary, this pathological survey demonstrated preservation of the cerebellar Purkinje cell layer even at 8 months of age, but progressive intracellular inclusions in liver, spleen and lung.
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Clinical course of wild-type, ASMKO and ASMKO/ChimTg mice
A cohort of wild-type, ASMKO and ASMKO/ChimTg mice of each gender was observed from birth for growth and weight changes and for symptoms of cerebellar dysfunction (e.g. body tremors and ataxia), which is characteristic of the lysosomal storage disease in ASMKO mice (19,20). Mice in all three groups appeared normal at birth and gained weight at similar rates up to 16 weeks of age (Fig. 8). By 10–12 weeks, as expected (19,20), the ASMKO pups started to display noticeable body tremors and an ataxic gait. The symptoms grew progressively worse thereafter and the mice became increasingly lethargic and unresponsive to touch. By 28 weeks, the mice demonstrated very little spontaneous movement and all were dead by 36–40 weeks. Both male and female ASMKO mice steadily lost weight from 16 weeks of age; at the time of death, the weight in the ASMKO mice had decreased to 50% of the level in wild-type mice (Fig. 8). In striking contrast, the overall appearance and behavior of the ASMKO/ChimTg-14 and -19 mice were indistinguishable from those of wild-type mice. Both lines of ASMKO/ChimTg mice remained as active as the wild-type mice throughout the 48–52 week observation period, there were no signs of body tremors or ataxia and none of the mice died by 52 weeks. The overall weight pattern of the ASMKO/ChimTg-19 mice was similar to that of wild-type mice throughout the observation period (Fig. 8). Thus, brain L-SMase activity as low as 11–18%, even in the face of visceral organ SM accumulation and progressive SM accumulation in the brain, dramatically improved the neurological deficit of ASMKO mice and the overall health and survival of these mice were indistinguishable from those of wild-type mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A critical issue in the treatment of the neurological forms of lysosomal storage diseases is how much intra-lysosomal hydrolase activity in the brain is necessary for clinical improvement (3). In the case of Niemann–Pick disease and other lysosomal storage disorders, attempts to address this issue in man by comparing lysosomal enzyme activity in cultured fibroblasts from patients with the absence or presence of neurological symptoms have yielded less than definitive results (2). Therapeutic studies with animal models have also left uncertainties regarding this critical issue because: (i) neurological correction in these studies relied on the uptake by neurons of soluble forms of acid hydrolases rather than expression in neurons themselves, and it has even been suggested that bone marrow-derived cells that enter the CNS after bone marrow transplantation may phagocytose storage material (29); (ii) the experimental conditions often used (e.g. high dose irradiation in bone marrow transplantation studies and injection of viral vectors in gene therapy experiments) may have led to disruption of the blood–brain barrier and/or infiltration of immune cells into the CNS; (iii) the enzyme delivery systems have often been transient due to the use of weekly or biweekly intravenous enzyme infusions, transplantation of short-lived cells into the CNS or promoter shutdown of viral vectors; and (iv) only small numbers of enzyme-secreting cells can be introduced into the CNS by bone marrow transplantation or direct intracerebral injection. Thus, the results have often been partial and difficult to interpret.
In the study reported herein, we have utilized a model of permanent intra-lysosomal enzyme replacement in the absence of soluble enzyme, radiation exposure or viral infection. The purpose of this study was not to develop a strategy that would be useful therapeutically in man but rather to determine in a more definitive manner the relationship between direct replacement of intra-lysosomal enzyme activity per se and neurological improvement and survival status. Our results indicate that in the case of ASMKO mice, a model of type A Niemann–Pick disease, brain L-SMase activity in the range 11–18% can lead to dramatic and long-lasting preservation of essentially normal CNS function, overall health and life span. In fact, given the visceral SM accumulation, these mice represent the first time that a germline animal model of a neurological storage disease (type A Niemann–Pick disease) has been made into a non-neurological form of the disease. As such, the mouse model created here has the major phenotype of type B Niemann–Pick disease, namely visceral SM accumulation in the absence of severe neurological disease, although actual type B disease in humans has partial deficiency of S-SMase and L-SMase rather than low-activity chimeric L-SMase plus complete absence of S-SMase.
Of interest, the degree of L-SMase activity achieved in the transgenic mice did not totally prevent SM accumulation in the brain, as demonstrated by the data with 8-month-old mice in Figure 5B. In fact, the brains of ASMKO/ChimTg mice had 8- to 11-fold more SM than those of wild-type mice at this time point. Despite this finding, the chimeric mice showed no signs of ataxia or tremors for their entire life span, which was equal to that of wild-type mice, and their cerebellar Purkinje cell layer remained intact at least up to 8 months of age. One possible explanation for this result is that total brain SMase and SM mass measurements do not accurately reflect these parameters in the area or areas of the brain most critical for clinical symptoms, which is presumably the cerebellum in general and the Purkinje cell layer in particular. Another explanation, however, is related to previous findings with Purkinje cells in culture. In these studies, ceramide and ceramide metabolites were shown to be essential for Purkinje cell growth, differentiation and survival, and treatment with SMase could promote these processes and prevent apoptosis (30,31). Thus, in the ASMKO/ChimTg mice, there may be enough SMase activity to generate ceramide for Purkinje cell survival but not enough to prevent SM accumulation over time; in this scenario, accumulation of SM per se would not be the important parameter.
A related issue to arise from our study is the relationship between tissue L-SMase activity and SM accumulation (Table 1 and Fig. 5). Conzelmann and Sandhoff (32) have presented a kinetic theory that takes into account both the residual enzyme activity and substrate concentration in the lysosome. When enzyme activity is low but above a certain threshold level, the increase in lysosomal substrate concentration can activate the residual enzyme towards Vmax so that substrate influx into lysosomes still equals substrate degradation, albeit at a higher steady-state of intra-lysosomal substrate concentration. When enzyme activity drops below this threshold, however, substrate degradation can no longer keep up with influx, resulting in massive intra-lysosomal substrate accumulation. Importantly, Leinekugel et al. (33) pointed out the limitations of studying this relationship in vitro using cell culture models. Indeed, although the fundamental principle of this hypothesis is undoubtedly correct [e.g. increased SM accumulation in ASMKO/ChimTg-19 versus ASMKO/ChimTg-14 kidney (see Results)], data from our in vivo model suggest that different tissues have different enzyme activity threshold values. For example, ASMKO/ChimTg-19 spleen had very low SMase activity (2.8% of wild-type), yet SM accumulation was relatively low. In contrast, ASMKO/ChimTg-19 liver had 12% of wild-type SMase activity yet had substantial SM accumulation.
We have been careful to document that the chimeric SMase resides in perinuclear compartments that contain the lysosomal enzyme cathepsin D. It is possible, however, that the trafficking route of the chimeric SMase to these lysosomes may not be identical to that of native L-SMase, which is targeted to lysosomes via vesicles containing the mannose 6-phosphate receptor. Although both mannose 6-phosphate receptors and lysosomal membrane proteins appear in trans-Golgi network-derived, clathrin-coated, AP-1-containing vesicles (34), these vesicles appear to be distinct from each other (35). In terms of trafficking pathways, hydrolases delivered to lysosomes via the mannose 6-phosphate receptor pathway, at least in many cell types, transiently pass through early endosomes (36–38). In contrast, Press et al. (38) have shown that trafficking of the rat homolog of Lamp1 to lysosomes in BHK cells largely bypasses early endosomes. Moreover, there are reports of lysosomal membrane proteins appearing transiently at the cell surface in certain cell types (39), which is not a site of lysosomal hydrolase delivery. Given these trafficking differences, it is theoretically possible that chimeric SMase, unlike native L-SMase, might have access to SM pools in the plasma membrane but not in early endosomes. The steady-state residence time of the enzyme in these organelles, however, may be too low to be functionally important. For example, with normal protein expression levels, only a very small proportion of lysosomal membrane proteins are at the cell surface (22,39). Moreover, it is likely that at least a portion of chimeric SMase molecules are mannose-phosphorylated and these molecules might traffic to lysosomes via the mannose 6-phosphate receptor system. Future trafficking studies will be needed to clarify these issues and to determine their relevance to organ physiology in the genetically engineered mice.
The approach used in this study applied fundamental principles of lysosomal protein targeting, heretofore demonstrated solely in cell culture studies, to genetically manipulate mice in vivo. This strategy could easily be applied to mouse models of other neurological forms of lysosomal storage disease to discern relationships between intra-lysosomal enzyme restoration per se and clinical parameters. Moreover, similar models lacking secretory hydrolase activity but with lysosomal enzyme activity that is closer to wild-type values could be extremely valuable in elucidating roles of secretory lysosomal hydrolases. For example, secretory SMase has been proposed to play important roles in atherogenesis (40,41) and possibly radiation- and lipopolysaccharide-induced endothelial apoptosis (42,43). Likewise, the secretory forms of other acid hydrolases have been proposed to play roles in tumor metastasis, sperm development and apoptosis (44–49). In fact, because peptide targeting motifs have been identified for most organelles, the strategy should be applicable to the in vivo investigation of other proteins with more than one intracellular destination.
In summary, we have utilized a novel transgenic/knockout strategy to demonstrate the direct relationship between permanent intra-lysosomal enzyme replacement and clinical outcome in a neurological form of a lysosomal disease. This is the first study demonstrating long-lasting preservation of neurological function in the absence of several confounding factors present in other models, such as replacement based on uptake of secretory enzymes or less well defined mechanisms and exposure to radiation and viruses. Thus, the findings in this report shed light on how much brain intra-lysosomal hydrolase activity is needed to prevent or correct neurological function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the ASM–LAMPI chimeric transgene
A schematic of the complete ASM–Lamp1 chimeric transgene is shown in Figure 1A. A short DNA sequence encoding the transmembrane domain and lysosomal targeting peptide of human Lamp1 was fused to the 3' end of the last exon of the murine ASM gene (mSM) and this chimera was placed between a 5' ß-actin promoter fragment and 3' SV40 polyadenylation cassette. To accomplish this construction, a 250 bp ASM–Lamp1 chimeric nucleotide sequence was created by PCR using human Lamp1 cDNA as template plus a sense ‘bridge’ primer, 5'-gcctgtggccaaggccactgttttgcctgatccccatcgctg-3' (Lamp1 sequence underlined) and an antisense primer, 5'-cagagaaaggaacagaggcccctgcagctg-3' (derived entirely from the Lamp1 sequence). The 250 bp PCR product, which was sequenced for verification, was cleaved with MscI and PstI and cloned into the 3' end of the last exon of the mouse ASM genomic sequence (plasmid clone pmSM in pBluescript II KS) to generate pmSM-Lp.
Next, to insert the promoter sequence, a 4.5 kb EcoRI–BamHI fragment containing the human ß-actin promoter sequence, which was derived from the pHß-Apr-1-neo plasmid (50), was cloned into pBluescript II KS to generate pß-actin. A 4.3 kb HindIII–XbaI fragment from pmSM-Lp (see above) was subcloned into the HindIII and XbaI sites of pß-actin to give pß-actin-mSM-Lp. To insert a final intron and polyadenylation sequence, a 0.7 kb XhoI–NcoI fragment containing the SV40 intron sequence and polyadenylation signal was isolated from pcDNAI (Invitrogen, Carlsbad, CA) and, after flushing the ends with Klenow fragment, the blunt ends were ligated into the EcoRV restriction site of pBluescript to create pSV40p/A. Finally, the 0.7 kb XbaI–NotI fragment from pSV40p/A was ligated into the XbaI and NotI sites of pß-actin-mSM-Lp to create the final transgene, pß-actin-mSM-Lp-p/A.
Generating pß-actin-mSM-Lp-p/A transgenic mice in the ASMKO background
A 9.5 kb KpnI–NotI fragment from pß-actin-mSM-Lp-p/A was separated from the vector backbone, purified by CsCl ultracentrifugation and microinjected into fertilized mouse eggs (C57BL/6J/CBA F1 background). The injected oocytes were then implanted into the oviducts of pseudopregnant female mice and the pups screened for presence of the pß-actin-mSM-Lp-p/A transgene by Southern analysis (see below). Two founders, labeled Tg14 and Tg19, were mated to ASMKO mice and transgenic pups heterozygous for the ASM locus were backcrossed with ASMKO mice to generate homozygous ASMKO mice containing the pß-actin-mSM-Lp-p/A chimeric transgene (ASMKO/ChimTg).
The screening strategy for identifying the ASMKO/ChimTg-19 line by Southern analysis using a full-length mouse ASM gene probe is shown in Figure 1B. When cut with BamHI and HindIII, the wild-type ASM gene generated a single 4.4 kb fragment (lane 1), the chimeric transgene a 15 kb fragment (lane 2) and the targeted ASM gene in ASMKO mice a 6.6 kb fragment (lane 3). Thus, heterozygous ASMKO mice containing the chimeric transgene were easily identifiable by the presence of all three bands on the Southern blot (lane 4). Similarly, ASMKO mice containing the chimeric transgene, which were generated by mating heterozygous ASMKO/ChimTg mice with ASMKO mice, were identified by the presence of the 6.6 and 15 kb bands and absence of the 4.4 kb band (lane 5).
SMase and SM mass assays
The sources of enzyme for the SMase assays were serum, tissue homogenates and the conditioned media or homogenates of peritoneal macrophages, which were cultured and prepared for enzyme assay as previously described (17,51). S-SMase and L-SMase activities were assayed as described by Schissel et al. (17). SM mass in mouse tissues was assayed using the method of Miranda et al. (28).
Immunofluorescence microscopy
Double-label immunofluorescence labeling of SMase and cathepsin D in macrophages was conducted using the cold methanol fixation/permeabilization procedure of Khelef et al. (52). To inhibit the interactions of antibodies with Fc receptors, mouse IgG2a and anti-mouse CD12/CD32 Fc receptor antibodies were added to all antibody incubations (52). The primary antibodies used were 5 µg/ml rabbit anti-SMase IgG (26) (provided by Drs Henry Lichtenstein and G. Andrew Keesler, formerly of Amgen, Boulder, CO) and 10 µg/ml goat anti-cathepsin D peptide IgG (E-19; Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibodies were 5 µg/ml Alexa-568-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) and 10 µg/ml Alexa-488-labeled donkey anti-goat IgG (Molecular Probes, Eugene, OR). The cells were observed with a Zeiss LSM 410 confocal microscope using an argon–krypton laser (488 and 568 nm excitation) and a 100x objective (NA 1.4). The images were processed with Adobe Photoshop software. Specificity for the anti-SMase antibody has been demonstrated by us recently (26) and verified using macrophages from ASMKO mice. Specificity for the anti-cathepsin D antibody was demonstrated by showing absence of specific signal when the antibody was pre-absorbed with the cathepsin D peptide immunogen. Control experiments using each of the various combinations of primary and secondary antibodies separately indicated no antibody cross-reactivity and no fluorophore crossover.
Histochemistry
Tissue samples for light microscopy were fixed in buffered 10% formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin (28).
Statistics
Unless otherwise indicated, results are given as means ± SEM (n = 3); absent error bars in the figures signify SEM values smaller than the graphic symbols.
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ACKNOWLEDGEMENTS |
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The authors thank Dr Stuart Kornfeld (Washington University, St Louis, MO) for providing the human Lamp1 cDNA. This study was supported by National Institutes of Health grants HL56984 (to I.T. and K.J.W.) and HD28607 (to E.H.S.), a grant (RR0071) from the National Center for Research Resources for the Mount Sinai General Clinical Research Center, a grant from the March of Dimes Birth Defects Foundation (to E.H.S.) and a research grant from Berlex Biosciences (to I.T.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 212 305 9430; Fax: +1 212 305 4834; Email: iat1@columbia.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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